人高活性血管基质层细胞快速分离及荧光标记的实验研究

目的探讨一种人高活性血管基质层细胞(stromal vascular fraction cells,SVFs)快速分离及荧光标记的有效方法,为SVFs用于临床奠定理论基础。方法取植皮手术患者自愿捐赠的腹部皮下脂肪组织,分别用0.065%、0.125%、0.185%3种浓度的Ⅰ型胶原酶消化,提取SVFs。收集细胞悬液进行细胞计数,锥虫蓝染色计算细胞成活率。用1’双十八烷-3,3,3’,3’-四甲基吲哚羧化青-高氯酸盐(1,1’-dioctadecyl-3,3,3’,3’-2-tetramethy-lindocyanine perchlorate,DiI)荧光标记0.125%浓度组SVFs,倒置荧光显微镜下观察标记情况;将DiI标记的SVFs接种培养,倒置显微镜观察细胞形态学变化,MTT法检测DiI标记前后细胞增殖能力。结果 0.065%、0.125%及0.185%浓度组SVFs细胞总数分别为(138.68±11.64)×104、(183.80±10.16)×104、(293.07±8.31)×104个,组间比较差异均有统计学意义(P<0.01);细胞成活率分别为91%±2%、90%±2%、81%±2%,0.065%浓度组和0.125%浓度组均显著高于0.185%浓度组(P<0.01),0.065%浓度组和0.125%浓度组比较差异无统计学意义(P=0.881)。倒置荧光显微镜观察示,0.125%浓度组细胞均可被DiI荧光标记,标记后的细胞膜完整,细胞质丰富,细胞形态正常,细胞核未染荧光;DiI标记的SVFs细胞悬液接种培养后,细胞能顺利贴壁及传代。MTT法检测示DiI标记前后SVFs细胞生长曲线相似,生长趋势吻合。结论 0.125%浓度的Ⅰ型胶原酶可有效消化脂肪中的胶原组织,获取大量SVFs,同时对细胞损伤较小,是理想的工作浓度;DiI可有效标记SVFs。

Experimental study on fluorescent labeling and optimization method of purifying human stromal vascular fraction cells

Objective To find a kind of simple and effective method for purifying and labeling stromal vascular fraction cells(SVFs) so as to provide a theoretical basis for clinical application of SVFs.Methods The subcutaneous adipose tissue were harvested form volunteers.The adipose tissue was digested with 0.065%,0.125%,and 0.185% type I collagenase,respectively.SVFs were harvested after digestion and counted.After trypan blue staining,the rate of viable cells was observed.SVFs was labeled by 1,1’-dioctadecyl-3,3,3’,3’-2-tetramethy-lindocyanine perchlorate(DiI).The uorescent labeling and growth was observed under an inverted uorescence microscope.MTT assay was used to detect cell proliferation.Results The number of SVFs was(138.68 ± 11.64) × 104,(183.80 ± 10.16) × 104,and(293.07 ± 8.31) × 104in 0.065% group,0.125% group,and 0.185% group,respectively,showing signi cant differences among 3 groups(P < 0.01).The rates of viable cells were 91% ± 2%,90% ± 2%,and 81% ± 2% in 0.065% group,0.125% group,and 0.185% group,respectively,and it was signi cantly higher in 0.065% group and 0.125% group than in 0.185% group(P < 0.01),but no signi cant di erence was found between 0.065% group and 0.125% group(P=0.881).Inverted uorescence microscope showed that the cell membranes could be labeled by DiI with intact cell membrane,abundant cytoplasm,and good shape,but nucleus could not labeled.SVFs labeled by DiI could be cultured successfully and maintained a normal form.MTT assay showed that similar curves of the cell growth were observed before and after DiI labeled to SVFs.Conclusion The optimal collagenase concentration for purifying SVFs is 0.125%.DiI is a kind of ideal fluorescent dye for SVFs.